This invention relates to the production of antimicrobial agents for use in therapy and to methods for identifying suitable antimicrobial agents.
It is now widely recognised that conventional antibiotics are becoming less effective in treating microbial infections due to the spread of resistant microbial strains. It is therefore important to develop new antimicrobial agents to treat infection.
An approach to identifying potential antimicrobial agents is to select a suitable target, the inhibition of which will result in the eradication of the microbe with minimum effects on the host.
An important requirement is that the target should be essential to the viability of the microbe, as often a microbe will have an alternative mechanism for carrying out the function of the target. For example, the microbe may utilise a different enzyme or a different pathway to by-pass the inhibition of the target.
In considering novel single targets (e.g. those not part of large assemblies such as ribosomes) an important consideration is whether the target, usually an enzyme, is unique to bacteria. If so, its properties and suitability for the design or discovery of useful inhibitors can be exploited without detrimental effects on the host. Other targets, such as dihydrofolate reductase (DHFR), are common to both bacterial and animal cells and therefore selective inhibition of the microorganism is essential if dose-dependent toxicity to the host is to be avoided. The antimicrobial drug trimethoprim is a useful example of a clinically-valuable DHFR inhibitor which competes with substrate (dihydrofolate) and is bound 50,000-fold more tightly by bacterial than mammalian forms of DHFR.
There are therefore several considerations that must be made when selecting a suitable target, and these considerations make the selection of a suitable target difficult.
Therefore, there is a recognised need for identifying and characterising suitable targets that may be useful in antimicrobial therapy. In particular, there is a need for identifying targets that may be used to screen compounds for antimicrobial activity which can be used to prevent, reduce or eradicate infections.
The present invention is based on the realisation that enzymes involved in co-enzyme A (CoA) synthesis may be suitable targets for antimicrobial or antiparasitic agents. In particular, it has been appreciated that the enzyme phosphopantetheine adenylyltransferase (PPAT) is a suitable target to inhibit the Coenzyme A synthetic pathway, thereby preventing bacterial growth. The suitability of PPAT as an antimicrobial or antiparasitic target is based, at least in part, on an appreciation that the differences in the PPAT enzyme in mammalian and bacterial systems may be exploited to provide selective inhibition of the bacterial form. In particular, the mammalian form exists as a bifunctional enzyme complex with dephospho-CoA kinase (dCoAk), whereas the bacterial form contains separate PPAT and dCoAk. Similar structural differences are found in yeast, fungi and parasitic forms of PPAT.
Therefore, the present invention relates to the use of bacterial or similar PPAT enzyme to select for antimicrobial agents. According to the present invention, a method for identifying antimicrobial agents, comprises the steps of:
(i) contacting a sample containing non-mammalian PPAT enzyme with a suitable substrate and a target compound under suitable conditions;
(ii) measuring the activity of the PPAT enzyme;
(iii) comparing the activity of the enzyme to that of a reference sample lacking the target compound; and
(iv) selecting those targets compounds that reduce the activity of the PPAT enzyme.
In a further bodiment, the present invention proposes the use of compounds capable of inhibiting the PPAT enzyme at a cellular or tissue concentration of less than 10 xcexcM in antimicrobial therapy. In particular, the compounds may be used in the treatment of infection by E. coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Neisseria meningitidis and Staphylococcus aureus, or any other pathogenic bacteria where PPAT activity is required for CoA synthesis.
According to a further embodiment, a substantially pure non-mammalian PPAT enzyme, or active fragment thereof, is used in a method for the identification of an antimicrobial or antiparasitic agent. Similarly, a gene encoding a non-mammalian PPAT enzyme may be used in a method for the identification of an antimicrobial or antiparasitic agent.
According to the present invention, methods for treating bacterial infection comprise selectively inhibiting the non-mammalian form of phosphopantetheine adenylyltransferase (PPAT). It has been recognised that this enzyme, also known as dephospho-CoA pyrophosphorylase, has particular characteristics which make it suitable as a target for inhibiting bacterial growth.
The term xe2x80x9cPPAT enzymexe2x80x9d refers to the complete catalytically-active PPAT enzyme, or to fragments of the enzyme that retain catalytic activity. The enzyme may be used in the methods of the present invention either immobilised on a solid support or in solution.
The term xe2x80x9cmicrobialxe2x80x9d relates to any microorganism that is capable of causing infection, e.g. bacterial, fungal and yeast. In particular the term relates to bacterial organisms, especially those implicated in infectious disease, for example, E. coli, Pseudomonas aeruginosa, Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Neisseria meningitidis and Staphylococcus aureus. 
PPAT enzymes may also be derived and purified from parasitic organisms which require the enzyme activity for CoA synthesis.
The term xe2x80x9ctarget compoundxe2x80x9d relates to any suitable agent that may be used as an inhibitor of PPAT activity. The compound may be biological, i.e. a protein or peptide that binds to and inhibits PPAT activity or alters the conformation of the enzyme in such a way as to block the substrate from the catalytic region, or may be a chemical compound. In either instance, the compound may be found naturally or may be synthesised. Suitable synthesis techniques will be apparent to those skilled in the art.
Possible target compounds may be chemically related to the natural substrates of PPAT, i.e. phosphopantetheine and ATP. Such substrates are converted to products at the catalytic centre of the enzyme, and so it is reasonable to assume that derivatives or analogues of such substrates may be identified which lead to selective inhibition of non-mammalian PPAT. In addition, compounds unrelated to the natural substrates may also be useful inhibitors. It is also envisaged that the inhibitors of PPAT activity may not act directly on the PPAT enzyme, but may instead act at the genetic level to block further production of PPAT.
The term xe2x80x9cactivityxe2x80x9d refers to the natural biological activity of the PPAT enzyme. Usually this activity takes the form of the transfer of the reactive adenylyl group from ATP to phosphopantetheine, yielding dephospho-CoA. However, enzyme reactions seldom proceed only in the forward direction, and the reverse direction may also be utilised for determining the activity of PPAT. Use of the reverse reaction may be beneficial as it is linked to the formation of ATP which may then be utilised in fluorescence or luminescence detection techniques, for example the ATP-dependent oxidation of luciferin by luciferase with the concomitant production of light. Other ways of measuring PPAT activity will be apparent to those skilled in the art and will be useful in the methods of the present invention.
The term xe2x80x9ctherapyxe2x80x9d refers to the treatment of diseases arising as a result of infection, not only by bacteria but also by fungi or parasites for which PPAT is required for the synthesis of CoA.
The term xe2x80x9cmedicamentxe2x80x9d refers to any suitable pharmaceutical composition. Specifically, it refers to a composition comprising the anti-infective agent in any suitable excipient or diluent, and also to different formulations required for different methods of administration. For example, the medicament may be formulated for oral administration, or may be formulated for intravenous administration.
Suitable excipients or diluents will be apparent to the skilled person, and the amount of antimicrobial agent present in the formulation can be easily determined, and will depend, in part, on the activity of the antimicrobial agent, and its side-effect profile.
The PPAT enzyme that is proposed for use in the present invention may be any microbial or parasitic PPAT enzyme. The existence of PPAT enzymes are known and are disclosed in, for example, Abiko et al, J. Biochem. (1967); 61:309 and Suzuki et al., J. Biochem. (1967); 62:642. As discussed previously, microbial PPAT is very different from mammalian PPAT, which is not proposed for use in the present invention, other than as a control for identifying inhibitory compounds that are selective for the microbial or parasitic form. Particularly preferred PPAT enzymes for use in the present invention include any gram-positive or gram-negative bacterial organism, E. coli, Pseudomonas aeruginosa, Streptoccoccus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Neisseria meningitidis, Staphylococcus aureus or other examples of pathogenic bacteria. The deduced amino acid sequence of the PPAT enzyme is well conserved in many different bacterial species, and so selecting an enzyme from one organism may permit an extrapolation of results to other organisms. However, it may be preferable for optimal therapeutic effect if the enzyme is selected from a particular bacterial species causing infection.
The PPAT enzyme may be purified from the organism using any conventional method. Suitable methods are known to those skilled in the art and include those methods disclosed in Worrall et al, Biochem. J. 1983; 215: 153-157. The purification may be carried out using protein purification techniques, e.g. ion-exchange chromatography and gel filtration techniques. The PPAT enzyme may also be produced by solid phase synthesis, or by recombinant DNA methods using the cloned PPAT gene or a fragment of this gene.
The PPAT gene is termed kdtB, and the sequence is available from DDBJ/EMBL/GenBank. Although the sequence is known, the function of the gene was thought initially to be linked to an aspect of the lipopolysacchanide assembly (Clementz and Raetz, J. Biol. Chem., 1991; 266:9687-9696), but has now been identified as PPAT, by the present inventors.